ARTICLE pubs.acs.org/biochemistry
Water in the Active Site of Ketosteroid Isomerase Philip Hanoian and Sharon Hammes-Schiffer* Department of Chemistry, 104 Chemistry Building, Pennsylvania State University, University Park, Pennsylvania 16802, United States
bS Supporting Information ABSTRACT: Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wildtype (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogenbonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogenbonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.
Δ5-3-Ketosteroid isomerase (KSI) is an extensively studied enzyme that serves as a useful model system for the study of the role of hydrogen bonding in enzyme catalysis. Two homologous bacterial forms of this enzyme from Pseudomonas putida (pKSI) and Commamonas testosteroni (tKSI) have been studied. In this paper, the residues are numbered according to pKSI. The structure of the active site residues in pKSI is depicted in Figure 1.1 KSI catalyzes the stereospecific isomerization of steroid substrates such as 5-androstene-3,17-dione (5-AND) and 5,10-estrene3,17-dione (5,10-EST).2 In this isomerization, Asp40 serves as a general base and subsequently as a general acid in two sequential proton transfer reactions, as illustrated in Figure 2 for the 5-AND substrate. The reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with Tyr16 and Asp103. The role of these hydrogen bonds in catalysis has been studied extensively using site-directed mutagenesis,35 and the properties of these hydrogen bonds have been probed using intermediate analogs such as equilenin (EQU) in conjunction with spectroscopy and X-ray crystallography.1,614 The dienolate intermediate forms of both steroid substrates and the anionic form of EQU are shown in Figure 3. The effects of the hydrogen bond-donating residues on the catalytic rate have been studied by site-directed mutagenesis using both the 5-AND and 5,10-EST substrates. The chemical steps are thought to be rate-limiting for the 5,10-EST substrate but not for the 5-AND substrate in wild-type (WT) KSI;15,16 however, the relative effect of mutation on the catalytic rate constant (kcat) is r 2011 American Chemical Society
generally similar for these two substrates.17 The Y16F mutation has been found to significantly influence KSI activity, reducing kcat by a factor of 103.2104.2.3,4,16 Mutations of less bulky residues (Y16T, Y16S, Y16A, and Y16G) were found to result in less significant reductions in kcat of ∼102.3.5 Each of these mutants was found to have a similar catalytic rate constant, regardless of the residue’s ability to serve as a hydrogen bond donor. In the same study, crystallographic data for the Y16S mutant (PDB entry 3IPT)5 showed additional electron density in the active site not attributable to any residue. This electron density was suggested to arise from the presence of disordered water molecules in the active site. According to this interpretation, water occupies the cavity created by the Y16S mutation and plays a role in catalysis as a hydrogen bond donor during the Y16S mutant enzymatic reaction. In contrast, the Y16F mutant was proposed to prevent the formation of these additional hydrogen bonds between water and the dienolate intermediate, thus conferring an apparently enhanced energetic penalty relative to the reference reaction in water. In the Y16F mutant, the additional Y32F/Y57F mutation was found to partially restore activity, a phenomenon termed “pseudoreversion”.4 This triple mutant species is significantly more active than the Y16F single mutant, with a reduction in kcat of ∼101.7 relative to that of WT KSI. Analysis of X-ray Received: May 6, 2011 Revised: June 27, 2011 Published: June 28, 2011 6689
dx.doi.org/10.1021/bi200703y | Biochemistry 2011, 50, 6689–6700
Biochemistry
ARTICLE
crystallographic data identified a small conformational change in the side chain of Y16F that was partially alleviated in the triple mutant. This conformational change was proposed to be the underlying structural cause of the pseudoreversion.4 Several studies have provided experimental insight into the degree of solvation in the active site of KSI and its mutants. Choi and co-workers used acrylamide quenching of the fluorescence of Trp120, which hydrogen bonds to the side chain carbonyl of Asp40, to probe the solvation of the active site of apo KSI.4 They observed a greater degree of quenching in WT KSI and the Y16F/ Y32F/Y57F (FFF) mutant than in the Y16F mutant. Relating these data to kinetics results, they proposed that a destabilizing structural change occurred in the Y16F mutant that was absent in WT and partially recovered by the triple phenylalanine mutation. The same study examined the effect of D2O on the reaction rate; they observed a small effect on the catalytic rate constant in WT KSI and the FFF mutant and a significant effect on the catalytic rate constant in the Y16F mutant. Mildvan and co-workers had previously examined the WT and Y16F mutant rates in D2O, concluding that the second step of the Y16F mutant reaction occurred only after dissociation of the intermediate (i.e., these authors proposed that this mutant followed a different mechanism).18 Recently, Kraut et al. compared the 19F NMR chemical shift of 2-fluoro-4nitrophenolate bound to the Y16F and Y16S mutants to the solution spectra in water and THF.5 The 19F chemical shift of 2-fluoro-4-nitrophenolate bound to the Y16S mutant was similar to that obtained in water, whereas the corresponding 19F chemical shift in the Y16F mutant was similar to that obtained in THF. These data were interpreted to suggest that the degree of solvation in the active site was considerably greater in the Y16S mutant than in the Y16F mutant.
The role of water in protein active sites has been discussed extensively in the literature.1922 Two previous theoretical studies have considered the presence of water in the KSI active site. Feierberg and Åqvist used empirical valence bond molecular dynamics (MD) simulations to evaluate the free energy profile of the isomerization reaction of 5-AND catalyzed by WT pKSI, both with and without a water molecule occupying a small cavity in the active site (red sphere in Figure 1).23 Mazumder et al. performed 1.5 ns equilibrium MD simulations of the WT tKSI enzymesubstrate and enzymeintermediate complexes of the 5-AND isomerization reaction.24 Water spontaneously entered the active site and occupied the position identified by Feierberg and Åqvist in the enzymesubstrate system but not in the enzyme intermediate system on the time scale of their simulations. More recently, a water molecule has been observed to occupy this site in high-resolution crystal structures of the D40N/D103N mutant with bound EQU (PDB entry 3FZW)10 and the D40N mutant with bound 2-fluorophenolate (PDB entry 3CPO).25 Furthermore, the presence of a hydrogen bond between a water molecule and the nitrile group of the D40N/M116C-CN mutant has been identified using 13C NMR and FTIR spectroscopies,26 supporting the presence of a water molecule within hydrogenbonding distance of the ligand oxygen. The objective of this work is to identify the number of water molecules in the active site of WT pKSI and several Tyr16 pKSI mutants and to elucidate the structural and dynamical properties of these water molecules. This information is not readily obtained with experimental methods. Our goal is to explore the hypotheses previously proposed to explain the available biochemical and structural data. To address these issues, we performed MD simulations on four pKSI species, WT, Y16F, Y16S, and FFF, each with the dienolate intermediate forms of 5-AND and 5,10-EST, as well as the intermediate analog EQU. The results support the hypothesis that several water molecules occupy the active site of the Y16S mutant and form hydrogen bonds to the dienolate intermediate oxygen during the enzymatic reaction. The simulations also indicate that the FFF mutant is similar to the Y16F mutant in that both mutants accommodate similar numbers of water
Figure 1. pKSI D40N active site with bound EQU from the 1OGX crystal structure, with the active site water molecule identified in the 3FZW crystal structure (pKSI D40N/D103N with bound EQU) shown as a red sphere. This water molecule was added by superimposition of the heavy atoms of EQU in both structures. The hydrogen-bonding network with OO distances of
